Background: Ambulatory assessment (AA) is increasingly recommended for assessing symptoms of posttraumatic stress disorder (PTSD). Previous AA studies provided new insights into the phenomenology of trauma-related memories, but also divergent findings. Notably, the range of trauma-related memories (a major target of psychotherapeutic interventions) reported in AA studies was as wide as 7.3 to 74.5 per week which might result from different methods used in these studies.Objective: We aimed at assessing the frequency of trauma-related memories in PTSD related to interpersonal violence and investigated whether this frequency is dependent upon the method.Method: For each patient trauma-related memories were assessed using two variants of smartphone-based AA: (1) Event-based sampling (EBS), i.e. participants entered data on each intrusive memory as it occurred; (2) Time-based sampling (TBS), i.e. participants reported the number of trauma-related memories they had experienced during the last two hours after they had been alerted by the smartphone. The numbers reported during the TBS-block were either analysed as reported by the participants or restricted to one per hour (rTBS). The impact of smartphone-assessments on trauma-related memories was assessed during a post-monitoring questionnaire.Results: While trauma-related memories were frequent across assessments, the methodology had a huge impact on the numbers: EBS (median = 7) and rTBS (median = 6) yielded significantly lower weekly numbers of intrusive trauma-related memories than TBS (median = 49). Accordingly, the possibility to report unrestricted numbers of trauma-related memories clearly impacted the results. The post-monitoring questionnaire identified another source for the divergent findings: while feeling disrupted by the smartphone-assessments was unrelated to the numbers reported during EBS, feeling disrupted was related to an increase of trauma-related memories during TBS and rTBS.Conclusions: The method clearly impacts the recorded number of trauma-related memories. Future research should clarify whether other variables (e.g. the subjective stress related to intrusive memories) are less dependent on the methodology. Data collection was complemented with a post-monitoring questionnaire about the potential impact of the assessments on trauma-related memories.2.2.1.Subjects were recruited between 2013 and 2015 from: (1) local psychotherapists in the centres, suburbs, and surroundings of four German cities: Mannheim, Ludwigshafen, Heidelberg, and Berlin; (2) from the waiting lists for psychotherapeutic treatment at the Central Institute of Mental Health (CIMH), Mannheim, and the Charité-University Medicine, Berlin; and (3) by flyers which were displayed at the CIMH and the Charité-University. As the study started before the introduction of the DSM-5, participants were eligible if they met the diagnostic criteria for PTSD according to DSM-IV  which was specifically programmed for this study. All other functions of a smartphone were disabled. For each participant the assessment period included both a TBS-block (seven days) and an EBS-block (three days), for details see The participants were provided with a smartphone (LG-P760) designated to be exclusively used for the purpose of the study. The assessments were recorded by a professional app . The low frequency of prompts during the TBS-block of one per day was chosen to avoid reactivity. During these prompts participants were asked whether they have had trauma-related thoughts or memories during the last two hours and (if so) how many distinct episodes of trauma-related memories or thoughts they have had. In addition, they were asked how many minutes were filled with these thoughts or memories during the last two hours. Current levels of aversive inner tension for both periods with and without trauma-related memories were assessed on a visual analogue scale ranging from 0–100. During the TBS-block, the smartphone rang once in the morning  during one day in order to assess intrusive memories that may have occurred during nighttime. Participants were instructed to consider only memories related to their index trauma . Trauma-related memories were defined according to the DSM-IV and included sensory memories and trauma-related thoughts. By collecting six daytime assessments from 10am till 8pm each covering two hours and the nighttime assessment covering the time from 8pm to 8am, the assessments covered the complete period of 24 hours of a day spanned across seven days. The sequence of those seven assessments was randomized. To avoid that participants would anticipate activity of the smartphone at a specific time, the randomized sequence of the timing of the assessment was concealed to the participants. The participants were only told the smartphone would ring no more than once a day between 8am and 8pm.In contrast, during the three days of the EBS-block participants were instructed to carry the smartphone with them all the time and to start a data entry whenever an intrusive memory has occurred.At the end of the study all participants were asked to fill out a short standardized post-monitoring questionnaire adapted from Ebner-Priemer and Sawitzki  which wan = 66 participants met all inclusion criteria and provided their written informed consent. The study was approved by the competent ethics committee (ID: 2013-536N-MA) and registered at the German Clinical Trials Register (ID: DRKS00005705).A total of 112 subjects met the inclusion criteria in the telephone interview and 83 agreed to be assessed with a diagnostic interview. Of the interviewed subjects, 2.2.For the purpose of comparing our results with the results of other studies, the recorded numbers of trauma-related memories in the EBS- and TBS-blocks were expressed as numbers per week. For the TBS-block two approaches for calculating the frequency of trauma-related memories were used: (1) in line with Pitman et al.  and Priep-values ≤ .05 (2-tailed) were considered statistically significant. Data analyses were carried out using SAS™ (v.9.4).Since the numbers of intrusive memories were typically skewed, the average numbers were primarily reported as medians. Non-parametric Friedman- and sign-tests were used to test the hypothesis that the frequency of trauma-related memories is dependent upon the method of assessment. Friedman-tests were used for a global comparison (EBS vs TBS vs rTBS); sign-tests were used for pairwise post-hoc comparisons. Wilcoxon signed rank tests were used to compare the levels of aversive inner tension: (1) for the 2h-periods assessed during the TBS-block which included at least one trauma-related memory vs the 2h-periods assessed during the TBS-block which included no trauma-related memories; and (2) for the 2h-periods assessed during the TBS-block which included at least one trauma-related memory vs the EBS-phase. Kolmogorov-Smirnov tests were used to compare the numbers of trauma-related memories in the subgroups of participants who did report a significant increase due to the smartphone to those who did not. 3.3.1.SD = 11.6, range: 18–61). Pursuant to the inclusion criteria, all participants had a diagnosis of PTSD and the index trauma was related to interpersonal violence. In 47.0% of the participants the index trauma had occurred below 18 years; in 53.0% the index trauma had occurred in adulthood. With respect to the index trauma the mean age at the beginning of childhood abuse was 7.1 years . The mean age of the occurrence of interpersonal violence in adulthood was 32.8 . The mean time elapsed since the end of the interpersonal violence index trauma was 27.3 years (SD = 12.8) for participants with childhood abuse and 2.5 years (SD = 4.9) for participants who experienced interpersonal violence in adulthood. Next to a PTSD diagnosis, the participants met on average 1.3 (range: 0–8) current Axis I diagnoses. The most prevalent co-occurring psychiatric disorders were Major Depressive Disorder (34.8%), Specific Phobia (16.7%), Panic Disorder (15.2%), Social Phobia (13.6%), Eating Disorders (10.6%), and Somatoform Pain Disorder (7.6%). In addition, 7.6% of the patients met the diagnostic criteria of BPD. One out of three participants (33.3%) reported a history of at least one suicide attempt. On average, the GAF score was 54.5 . The mean scores for the self-ratings scales to assess psychopathology were as follows: DTS total score: 83.2 ; BSL-23 mean score: 1.6 , and BDI-II total score: 35.9 . The mean scores for sexual and physical abuse during childhood/adolescence as assessed from the respective scores of the CTQ were 10.9  and 9.9 , respectively. The majority of participants were regularly employed (58.7%); one out of three participants was either unemployed (17.5%) or on general disability pension (17.5%); the remaining four participants (6.3%) were in vocational training. Most of the participants (80.0%) were currently in psychotherapeutic treatment but without exposure elements .A total of 66 subjects  participated in the study. On average participants were 38.9 years old (3.2.3.2.1.SD = 14.1). The total number of trauma-related memories during the three days of EBS-assessments ranged from 0 to 13 which corresponds to a total of 0 to 30.3 intrusive memories in one week .During the three successive days of EBS, 83.0% of the participants reported at least one trauma-related memory. If more than one trauma-related memory during the EBS-assessments was reported, the median time between two reported memories was 15 hours . This corresponds to a median number of 49 trauma-related memories per week . When restricting the count of trauma-related memories during the TBS-block to one per hour (rTBS), the number of intrusive memories in one week was 1–26 .During the TBS phase a total of 80.2% of the assessments were answered, speaking of an overall good compliance. The missing data entries were due to technical problems (e.g. running out of battery power) and/or incomplete data entry. During the seven TBS-assessments covering 24h, 87.2% of the participants reported at least one trauma-related memory. During the six 2h-assessments covering the daytime from 8am to 8pm, 85.1% of the participants reported at least one trauma-related memory. For the night (i.e. from 8pm to 8am), this percentage was 63.8%. The total number of trauma-related memories reported during the seven TBS-assessments covering 24h ranged from 0 to 59 . Post-hoc tests indicated that the numbers were similar for EBS and rTBS , but differed significantly between EBS and TBS (p < .001) and between rTBS and TBS (p < .001).As illustrated in 3.3.SD = 26.5, range: 0–95) and 2h-periods which included at least one trauma-related memory . The large difference (42.3 vs 64.9) was statistically significant  and indicates that a high level of distress is related to trauma-related memories. During the EBS-phase the mean level of aversive inner tension was somewhat higher  and differed significantly from the 2h-periods assessed during the TBS-phase which included at least one trauma-related memory .During all assessments participants were asked about the current level of aversive inner tension. During the TBS-block the mean levels of aversive inner tension clearly differed between 2h-periods which were free of trauma-related memories . This corresponds to a mean of 11.4% of daytime.If trauma-related memories during a daytime assessment within the TBS-block were reported, the participant was automatically asked about the duration of these memories. When summing up the duration of intrusive memories during daytime assessments of the TBS-block, the participants were preoccupied by intrusive memories for a median time of 63 minutes during the 12 hours covered by the daytime assessments  and the rTBS-phase . However, during the EBS-phase, both subgroups reported the same median numbers of trauma-related memories . Accordingly, the prompts issued by the smartphone during the TBS-phase or the anticipation of these prompts might have induced trauma-related memories in a subgroup of participants.In the post-monitoring questionnaire which was administered at the end of the study, 14.5% of the participants reported that the time with the study-related smartphone was ‘as usual’. The percentages for the categories ‘nearly as usual’, ‘somewhat different’, and ‘completely different’ were 23.6%, 36.4%, and 25.5%, respectively. With regard to a possible impact of the smartphone assessment on the frequency of trauma-related memories, 78.2% of the participants reported that the smartphone did not significantly increase the frequency of trauma-related memories . However, 21.8% reported a ‘significant increase’ of trauma-related memories due to the study-related smartphone. When comparing the subgroups of participants who did report a significant increase due to the smartphone to those who did not, there was a difference in the median of the recorded numbers for both the unrestricted TBS-phase  restricted to one per hour (rTBS). A post-monitoring questionnaire was used to ask the participants to which extent the smartphone assessments might have altered their trauma-related memories during the study period.In our study, the weekly numbers of trauma-related memories were similar for the EBS- and rTBS-assessments , but much higher for the unrestricted TBS-assessment (median = 49). Accordingly, the possibility to report unrestricted numbers of trauma-related memories for short periods apparently had a large impact on the results. Data from the post-monitoring questionnaire revealed another source possibly accounting for divergent results between EBS- and TBS-assessments: while feeling disrupted by carrying the study-related smartphone and by being assessed with this smartphone was essentially unrelated to the numbers reported during EBS, feeling disrupted was related to a marked increase of trauma-related memories during TBS and rTBS.The study at hand confirms previous AA studies in that the number of trauma-related memories during everyday life typically exceeds the sensitive range of widely used paper-and-pencil instruments. This finding also holds true when restricting the number of reportable trauma-related memories to one per hour during the data analyses. The extremely high numbers from unrestricted TBS in our study further indicate that a substantial share of participants with PTSD after interpersonal violence experience episodes of traumatic memories very frequently. These high numbers are in line with Priebe et al.  and exteThe systematic manipulation of the assessment methodology in a within-subject design allows attributing the differences in EBS, TBS, and rTBS numbers to the methodology. As such, the data shed light on the discrepant numbers observed in previous AA-studies. Although the limited number of previous studies precludes firm conclusions, our data are in line with the observation that studies relying on TBS  partially addresses this point, the numbers calculated for rTBS tend to underestimate the numbers of trauma-related memories occurring at high frequencies. Furthermore, the numbers calculated for rTBS do not reflect the clinical phenomenon that PTSD-patients may sometimes feel deluged with trauma-related memories. As discussed above, EBS-assessments might have been subject to systematic underreporting as missed data entries were interpreted as ‘absence of trauma-related memory’ during the EBS-phase. This is a limitation inherent to the sampling scheme. On the other hand, in comparison to TBS and rTBS EBS tended to be more robust against biases resulting from feeling disturbed by using the study-related smartphone. Taken together, we are inclined to think that EBS-assessments provide a conservative and robust estimate for the number of trauma-related memories, at the expense of sensitivity. None of the currently used AA-methods for assessing intrusive memories allows for an easy interpretation. The exact method of assessment has always to be borne in mind when interpreting the numbers. A further limitation of our study relates to the high level of psychopathology and to the low level of global functioning in our sample. Accordingly, it is impossible to know whether the observed numbers of trauma-related memories can be generalized beyond the group of highly symptomatic PTSD related to interpersonal violence.However, when considering the results of the post-monitoring questionnaire and several counter-biases, we are inclined to think that the essence of some of the findings is not challenged. Our study confirmed that the average number of distressing memories in subjects with PTSD related to interpersonal violence is outside the sensitive range of traditional assessment instruments. This raises the question whether psychotherapeutic research should be complemented by more sensitive tools to capture intrusive memories than traditional paper-and-pencil instruments. For example, the widely used Posttraumatic Diagnostic Scale (PDS-5; Foa et al.,


Strand displacement in heterodimeric coiled coils follows a competitive binding mechanism and can be predicted by the ratio of KD values. de novo design, the parallel heterodimeric coiled coil is most frequently used in bioinspired applications and chemical biology in general. This is due to the straightforward sequence-to-structure relationships, which it has in common with all coiled-coil motifs, and the heterospecificity, which allows control of association. Whereas much focus was laid on designing orthogonal coiled coils, systematic studies on controlling association, for instance by strand displacement, are rare. As a contribution to the design of dynamic coiled-coil-based systems, we studied the strand-displacement mechanism in obligate heterodimeric coiled coils to investigate the suitability of the dissociation constants (KD) as parameters for the prediction of the outcome of strand-displacement reactions. We use two sets of heterodimeric coiled coils, the previously reported N-AxBy and the newly characterized C-AxBy. Both comprise KD values in the μM to sub-nM regime. Strand displacement is explored by CD titration and a FRET-based kinetic assay and is proved to be an equilibrium reaction with half-lifes from a few seconds up to minutes. We could fit the displacement data by a competitive binding model, giving rate constants and overall affinities of the underlying association and dissociation reactions. The overall affinities correlate well with the ratios of KD values determined by CD-thermal denaturation experiments and, hence, support the dissociative mechanism of strand displacement in heterodimeric coiled coils. From the results of more than 100 different displacement reactions we are able to classify three categories of overall affinities, which allow for easy prediction of the equilibrium of strand displacement in two competing heterodimeric coiled coils.Among the protein folding motifs, which are accessible by  De novo designed protein folding motifs have been increasingly applied in the design of bioinspired systems or biomaterials from the bottom-up.abcdefg register – significantly facilitates peptide design.e.g. acyl-transfer reactions,28Among the available coiled-coil assemblies the heterodimeric coiled coil has gained significant scientific attention. The heterogeneity of the two coil strands adds a control element to the assembly, which makes it an ideal simplified model for protein–protein interactions.e and g positions of one coil strand and negatively charged glutamic acid residues (Glu) to the e and g positions of the other coil strand to induce heterospecificity.a positions of a central coiled-coil heptad are incorporated as an additional design feature to improve the dimer specificity and to stabilize the parallel orientation.The simplest rules to design a parallel heterodimeric coiled coil include an isoleucine–leucine core to direct dimerization,xBy, was presented, in which the strength of coiled-coil interaction was modulated by the lengths of the individual coil strands.a position was vital for enabling the formation of distinct coiled-coil assemblies, in which the individual strands varied in lengths of 3, 3.5 and 4 heptads, truncated from the N-terminal end. Combination resulted in a set of nine heterodimeric coiled coils with dissociation constants in the μM to sub-nM regime.Recently, a set of heterodimeric coiled coils, AKD values within the AxBy set of heterodimeric coiled coils should open new routes to the design of dynamic functional systems based on strand displacement. Indeed, KD values have often been discussed as parameters to assess such displacement events in heterodimeric coiled coils.KD-value differences would be desirable to facilitate designs. Herein, we present an extensive study of strand displacement in AxBy peptides, which, for clarity reasons, we dub N-AxBy, and a similar set of heterodimeric coiled coils, C-AxBy, in which the individual peptides are truncated from the C-terminal end. We aim to understand the mechanism of strand displacement in heterodimeric coiled coils by performing CD-titration experiments and a FRET-based kinetic strand-displacement assay. This leads to an easy-to-use rule of thumb to predict outcomes of strand displacements purely based on KD values.The range of 2HPO4, 1.8 mM K2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). Peptide concentrations were determined by UV-absorbance at 280 nm (ε280(Trp) = 5690 mol–1 cm–1, ε280(Tyr) = 1280 mol–1 cm–1), and, alternatively, at 214 nm using a NanoDrop 2000 spectrophotometer from Thermo Scientific. Extinction coefficients at 214 nm were calculated according to Kuipers et al.50Fmoc-protected amino acids were purchased from GL Biochem LTD . DIC and Oxyma Pure were obtained from Iris Biotech GmbH . The H-Rink Amide-ChemMatrix® resin was acquired from Sigma Aldrich . DMF used for peptide synthesis was supplied by Fisher Scientific  and was of peptide grade quality. Acetonitrile used for HPLC was supplied by Fisher Scientific  with HPLC grade quality. Water used for HPLC and reactions was obtained by purifying deionized water with the purification device Simplicity from Millipore. All other reagents were supplied by Sigma Aldrich , Thermo Fisher Scientific , VWR International  and Carl Roth . All reagents were of synthesis grade quality and were used as supplied. Unless otherwise stated, biophysical measurements were performed in phosphate buffered saline ) for Fmoc-removal. N-Acetylation of the peptides was carried out by equilibrating the peptide resin with 5 mL of acetic acid anhydride/pyridine (1 : 9 (v/v)) for 5 min at room temperature. Acidic cleavage from the resin was achieved by a treatment with a mixture of trifluoroacetic acid (TFA)/triisopropylsilane/water (90 : 5 : 5 (v/v/v), 3 h). The resin was washed with additional TFA (5 mL), and the combined TFA fractions were concentrated to a third of the initial volume under a flow of nitrogen. The crude peptide was then precipitated from cold diethylether (40 mL) and isolated by centrifugation and decantation of the supernatant. The precipitate was washed twice with ice-cold diethylether and subsequently dissolved in 5 mL of a 1 : 1 (v/v) mixture of acetonitrile and water and then freeze-dried to give a fine white solid.The peptide amides were synthesized on a H-Rink Amide-ChemMatrix® resin on a 0.1 mmol scale on a Liberty Blue CEM microwave-assisted peptide synthesizer. The synthesis was conducted –1 and 3 mL min–1, respectively, and a Nucleodur 100-5-C18  reversed-phase column from Macherey-Nagel for preparative HPLC and a Nucleodur 100-5-C18 ec,  reversed-phase column from Macherey-Nagel for semi-preparative HPLC. Linear gradients of water and acetonitrile  of 30–60% buffer B over 30 min for A peptides and 20–50% buffer B over 30 min for B peptides were used for purification. Chromatograms were monitored at 220 nm wavelengths.Peptides were purified by preparative and semi-preparative reversed-phase HPLC using a JASCO chromatography system  at flow rates of 10 mL min–1. For peptide characterization a linear gradient of water and acetonitrile  run from 20–80% buffer B over 20 min for the A peptides and 10–70% buffer B over 20 min for the B peptides were used. Chromatograms were monitored at 220 nm wavelengths.The peptides were characterized by mass spectrometry on a Bruker Autoflex Speed MALDI-TOF mass spectrometer operating in positive-ion reflector mode (matrix: α-cyano-4-hydroxycinnamic acid (CHCA), external calibration). Analytical HPLC measurements were performed using a JASCO chromatography system  and a Nucleodur 100-5-C18  reversed-phase column from Macherey-Nagel at a flow rate of 1 mL min–1 scanning speed. CD-thermal-denaturation experiments were performed by heating from 5 to 95 °C at a heat rate of 40 °C h–1. The CD signal at 222 nm was recorded at 1 °C intervals . The midpoints of the thermal denaturation curves (Tm) were determined from the second derivative of the variable temperature slope. Equilibrium dissociation constants (KD) were determined using FitDis! 1.0.51CD spectra and CD thermal-denaturation profiles were recorded on a JASCO J-1500 CD spectrometer, which was equipped with a JASCO PTC-510 temperature measuring unit. CD spectra were measured at 50 μM peptide concentration in PBS at 20 °C in 1 mm quartz cuvettes from Starna at 50 nm min–1 scanning speed. For background correction the coil peptides were measured individually at 25 μM and 50 μM concentration. To process the mixing experiments, the obtained CD spectra were corrected by the CD spectra of the individual peptide, which was displaced in the experiment.Coiled-coil peptides were mixed in a 1 : 1 ratio in PBS and in 50 μM concentration of the individual coil peptides. After addition of the competitor peptide in half-equimolar and equimolar amounts, CD spectra were recorded at 20 °C in 1 mm quartz cuvettes from Starna at 50 nm min–1, 300 μL per well) for 2 h at 25 °C. Afterwards, the well plates were washed three times with 100 μL PBS. The fluorescently labelled heterodimeric coiled coils  upon addition of equimolar amounts of a non-labelled competitor peptide (Pcomp). To obtain minimum fluorescence (Fmin), another 50 μM Pcomp were added. The obtained data was baseline-corrected by Fmin, normalized to Fmax and fit to a single exponential decay function using OriginPro 8.5G (eqn (1)),Feq is the fluorescence at equilibrium, A the pre-exponential factor and kobs the observed rate constant. Half-lifes were calculated according to eqn (2).Strand-displacement experiments were performed as triple measurements and monitored over 60 s as a decrease of the FRET-acceptor fluorescence  and Fmin was assigned to full strand displacement in AB (15 μM APcomp or PcompB and 15 μM A or B) to ABcomp or AcompB, respectively. Same strand-displacement kinetics for coiled coils of the same lengths of the individual strands were presumed whether labelled or unlabelled. Fitting of displacement of A or B from the initial AB complex and re-association, gave relative rate constants k1 to k4 from which the overall affinities (k1k4/k2k3) were calculated.To create the input files for DynaFit, kinetic fluorescence data was converted into concentration of free A or B in the time course of the experiment and competitive reformation of the AB coiled coil. KD values of coiled coils. Recently, a set of heterodimeric coiled coils was presented that covered dissociation constants from micromolar to sub-nanomolar concentration  spectroscopy. CD spectra of all nine possible combinations of heterodimeric coiled coils show 58 to 85% α-helical structure displayed by the characteristic strong minima at 208 and 222 nm , and analyzed the peptide mixture by CD spectroscopy. We determined mixtures with Pcomp added in half-equimolar and equimolar amounts, respectively. In 3.5 to N-A4B3 and v.v.  titand v.v. , (c) titand v.v. , and (d)and v.v. . The obtAs clearly documented in KD values of about two magnitudes; hence, the driving force of a strand displacement in favour for the more stable coiled-coil pair is high. However, we investigated all possible coiled-coil combinations within N-AxBy and C-AxBy, respectively, and found that displacement of shorter coil strands by longer competing peptides is the most frequent scenario. Nonetheless, if the KD values were very similar, the mixing experiments gave more intense CD signals, compared to the CD spectra of the two competing heterodimeric coiled-coil pairs , and normalized by maximum FRET fluorescence Fmax. In case of partial or complete strand displacement, a decrease of the FRET signal was observed approaching a plateau in most of the measurements. This is explained by dissociation of the preformed labelled coiled coil and re-association to a mixture of heterodimeric coiled coils depending on the rates of dissociation and association of the respective coiled-coil assemblies. The kinetic data were fit well by a single exponential model (see ESIkobs) from 0.01 s–1 to 0.1 s–1 and respective half-lifes from 7 s to 70 s  were calculated (Tables S7 and S8KD values obtained from CD thermal denaturation curves (KD values of the two competing coiled coils should give the equilibrium concentration and therefore, should be suitable to predict the outcome of a displacement reaction.The kinetic data from the FRET assay revealed an equilibrium reaction for strand displacement in heterodimeric coiled coils, which is interpreted as a competition of Pn curves . This su3.5B3.5-B4 (KD values can be applied as a reliable rule-of-thumb to assess strand-displacement equilibria in any competing heterodimeric coiled coils, if the assemblies are non-promiscuously heterodimeric.We performed more than 100 displacement reactions, from which we were able to deduce an assignment of the obtained values. In 5B3.5-B4  the posi5B3.5-B4  or on th5B3.5-B4 . If this5B3.5-B4 , overallde novo designed parallel heterodimeric coiled coils. We investigated two sets of well-characterized heterodimeric coiled coils, which exhibit dissociation constants from the μM to the sub-nM regime. Peptide design is based on Hodges EK peptides,a position to increase the specificity for parallel-oriented dimeric assemblies. Furthermore, this additional design feature allows formation of defined coiled-coil assemblies with variable chain lengths, a key-requirement for easy modulation of the strength of coiled-coil association. Design and biophysical characterization of the N-AxBy peptides have been previously reported.KD values was achieved by truncating the individual coiled-coil strands from the N-terminal end. We expanded the existing set of well-characterized heterodimeric coiled coils by adding C-AxBy, which is based on the same design rules as N-AxBy, except the fine-tuning of the strength of association is achieved by truncating the individual coil strands from the C-terminal end. We consider C-AxBy as a useful addition to the coiled-coil toolkit as blunt-ended N-termini facilitate applications, in which N-terminal modified coiled coils are involved. C-AxBy peptides were characterized by CD spectroscopy, and KD values were determined from CD-thermal-denaturation profiles. Although the resulting assemblies showed an overall slightly reduced stability compared to N-AxBy the determined KD values covered a similar range of concentrations.In summary, we have presented a thorough study on single strand-displacement kinetics in KD values of the two competing assemblies gave inconclusive CD data. Using a FRET-based assay, we characterized strand displacements in our sets of heterodimeric coiled coils as equilibrium reactions with half-lifes of about 7 to 70 s. The kinetic data were fit well by a competitive binding model from which the rate constants of association and dissociation of the competing coiled-coil assemblies, and hence, the overall affinities were obtained. The overall affinities correlate well with the respective ratio of KD values obtained from CD data supporting the competitive binding mechanism of the strand displacement in heterodimeric coiled coils.We studied strand displacement in both sets of heterodimeric coiled coils. CD-titration experiments indicated a preference for the formation of the thermodynamically more stable coiled coil in most of the studied combinations. However, coiled-coil mixtures with similar KD values can be used to predict the outcome of a strand-displacement reaction. Based on the results of 108 possible strand-displacement reactions, we were able to correlate the ratio of KD values to the position of the strand-displacement equilibrium and to classify three categories: KD ratios of above 10.0 correlate with very slow to no strand displacement, whereas ratios below 0.1 indicate complete displacement reactions. With ratios of KD values between 10.0 and 0.1 the formation of coiled-coil mixtures is highly probable.The ratio of KD values of coiled coils are known and discussed as potential parameters to predict strand displacement.KD differences and strand displacement;KD values of the competing coiled coils as parameter to reliably predict the outcome of a strand-displacement reaction without further kinetic experiments. We believe that the abovementioned rule of thumb is easy to adapt to other sets of heterodimeric coiled coils based on similar designs, and hence, will facilitate future designs of dynamic coiled-coil-based systems.Our aim was to find a reliable rule to assess strand displacement in heterodimeric coiled coils. Very often the There are no conflicts to declare.Supplementary informationClick here for additional data file.


The incidence of rheumatoid arthritis (RA) has been reported to be correlated with a disorder of immunregulation. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) play an important role in regulating the local immune microenvironment. However, the potential mechanism of RA-FLS in regulating the immnue response is not clearly understood. In this study, we demonstrated that the expression of HIF-1α was significantly up-regulated in rheumatoid arthritis tissue which indicated that the hypoxia condition in the microenvironment. We also observed that RA-FLSs demonstrated the potential to up-regulate immune activation. Meanwhile, the level of autophagy increased in RA-FLSs compared with control group. Besides that, the expression of IL-6 was up-regulated not only in RA-FLSs but also in the fibroblasts that treated with hypoxia condition. Accordingly, we found that autophagy inhibitiors could effectively inhibit the immune activation function of RA-FLSs medicated by IL-6. Taken together, the results we demonstrated above indicated that the hypoxia microenvironment could effectively induce the incidence of autophagy and then lead to the immune activation function of RA-FLSs medicated by IL-6. Rheumatoid arthritis (RA) is an autoimmune-mediated chronic inflammatory joint disease which affects approximately 1% of the population with disability and decreased quality of life in the end-stage . Many inFibroblast-like synoviocytes (FLS), located in the synovial tissue and strong correlation with the progress of RA, has drawn many researchers’ attentions , 9. MeanFormer studies indicated that inflammatory-related tissue damage can lead to local blood supply deficiency which can form a hypoxic microenvironment . It has In our present study, we aim to observe the effect of RA-FLS immune regulating function influenced by hypoxic microenvironment and further explore the potential mechanism. The result demonstrated that hypoxic stress can obviously increase the autophagy of RA-PLSs and inhibition the autophagy can significantly down-regulated the immune activation function of RA-FLSs. Furthermore, we found that the mechanism of autophagy increased by hypoxic stress enhanced the immune activation function via up-regulated the expression of IL-6.Firstly, we constructed the animal model of rheumatoid arthritis. In the tissues of rheumatoid arthritis, we could observe that the expression of HIF-1α was significantly up-regulated, which indicated that the hypoxia condition in the microenvironment Figure . BesidesRA-FLSs were isolated from rheumatoid arthritis tissues. Then the effect of RA-FLSs on the cell viability and apoptosis of lymphocytes was detected through cultivated with supernate of RA-FLSs. As shown in Figure It has been reported that hypoxic stress could effectively induce the incidence of autophagy –17. ThenIn order to confirm the role autophagy in the immune activation function of RA-FLSs, we used autophagy inhibitor 3-methyladenine (3-MA) and Chloroquine (CQ) to inhibit the autophagy in RA-FLSs. The results showed that the enhancement of RA-FLSs on the cell viability of lymphocytes was significantly decreased by 3-MA and CQ Figure . MeanwhiIt has been reported that IL-6 is a key immune factor which is up-regulated in the tissue of RA. Then we assess the secretion of IL-6 in RA-FLSs and N-FLSs by ELISA. As shown in Figure We have demonstrated that hypoxia condition could induce the incidence of autophagy, meanwhile it also lead to up-regulation of IL-6 in RA-FLSs. We employed autophagy inhibitor 3-MA and CQ to confirm the role of autophagy in up-regulating the expression of IL-6 not only in N-FLSs treated with hypoxia condition but also in RA-FLSs. The results demonstrated that when the autophagy was inhibited, hypoxia condition could not enhance the expression of IL-6 in N-FLSs Figure . As showIn our study, we showed the effect of immune activation function influenced by hypoxic condition in RA-FLSs and explored the possible mechanisms. Firstly, we found the high expression of HIF-1α by IHC in the animal with rheumatoid arthritis which indicated the incidence of rheumatoid arthritis is associated with hypoxia microenvironment. Subsequently, we separated the RA-FLS and healthy FLS from rheumatoid arthritis tissues and normal and then treated to T cells by co-culture. By detecting the cell viability and apoptosis of lymphocytes, we found that RA-FLS could obviously enhance the cell viability and decreased the apoptosis by comparing to healthy FLS-treated. Moreover, compared to control group, the autophagy of RA-PLS was significantly up-regulated by analyzing the expression of LC3 and p62 which was cultured in hypoxic condition. To further demonstrated the relationship between autophagy and immune activation function, autophagy inhibitors (3-MA and CQ) were used and the results indicated that inhibition of autophagy often accompany with down-regulation of immune activation function in RA-FLS. Furthermore, ELISA was performed and we observed that the secretion of IL-6 in RA-FLSs treated with hypoxic condition was obviously up-regulated. Additionally, inhibition of autophagy by 3-MA and CQ could effectively block the up-regulation of IL-6 caused by hypoxia condition, which demonstrated that RA-FLS enhanced immune activation function in hypoxic condition via increased autophagy-mediate the expression of IL-6.Rheumatoid arthritis is a chronic inflammatory and complex autoimmune disease . NowadayFormer studies indicated that inflammatory-related tissue damage can lead to local blood supply deficiency which can form a hypoxic microenvironment , 25. Hyp2. The 1% O2 condition was used as hypoxia condition. Chloroquine  and 3-methyladenine  were purchased from Sigma-Aldrich.The FLS cells were primary isolated from synovial tissues and cultured in Dulbecco's modified Eagle's medium (DMEM)  with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% COAll procedures involving animals were performed in accordance with the institutional animal welfare guidelines of Tongji University. Subcutaneous implantation of RA-FLS cells was performed in armpit areas of nude mice. Mice were examined three times per week.5cells/well) were cultured in 6-well plate. According to the manufacturer's instruction , PI/Annexin V-FITC assay was used to examine the apoptosis of the cells by flow cytometry. The lymphocytes were collected and washed with ice cold PBS three times. Then the cells were incubated in 300 μL of 1×binding buffer containing 5 μL PI and 5 μL Annexin V in dark at room temperature for 30 min. After incubation, the apoptosis of the cells was detected by BD FACS Calibur flow cytometer . The double positive cells (PI and Annexin V positive) were concerned as apoptotic cells [Cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The lymphocytes were seeded in 96-well plates (5000 cells per well). After 72h culturing, CCK-8 (10μl) was added in each well. The plate was then incubated at 37°C for another 2 hours. The optical density (OD) value at 450nm of each well was examined by spectrophotometer. All determinations were carried out in sextuplicate. The lymphocytes  and cultivated in 37°C, 15min after centrifuging. Sodium hydroxide (NaOH) then was added and strewed 3min for detecting the OD values in 440nm wave. The content of lactic acid was calculated according to the stand samples. The procedures were performed according to the manuscript of lactic acid testing kit .Total mRNA was isolated from synovial tissues or the cells by using Trizol reagent according to the protocol offered by the manufacturer. The expression of genes was determined by real-time PCR using the SYBR Green Master Mix Kit. The sequences of the primers were listed in Table All treated cells were washed with PBS and lysed by RIPA and PMSF at a ratio of 100:1 to obtain the total protein for Western blot. Equal amount of proteins was separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane. After transferring, the polyvinylidene fluoride membrane was blocked in 5% fat-free milk/1 × TBS/0.1% Tween-20 for 1 h at room temperature and then incubated with primary antibodies with gentle agitation overnight at 4°C. And then, the membrane was washed with 1 × TBS/0.1% Tween-20 before incubated with the secondary antibody according to the species of primary antibody for 1 h at room temperature. Immunoblots were performed by using the BeyoECL Plus substrate system (Beyotime), followed by washing with 1 × TBS/0.1% Tween-20. Western blot analyzed of LC3A/B (Abcam ab128025) and GAPDH .The shRNA sequences of genes were designed by using Oligoengine software and verified by nucleotide BLAST searches. According to the protocol from the manufacturer, the cells were plated in 6-well plates overnight and then Fugene HD transfection reagent (Roche) were used to transfect GFP-shRNA expression plasmids into the cells. A scrambled sequence was used as a negative control. At the end of each experiment, cells were observed by fluorescence microscope and/or Western Blot to determine transfection efficiency. The experiments were conducted in triplicate.The paraffin-embedded tissues were cut into 4-μm-thick slides and were deparaffinized and rehydrated. The slides were blocked with 10% normal goat serum, then rinsed in PBS and incubated for with affinity-purified antibody (HIF-1α) in 2% normal goat serum in PBS for overnight at 4°C. Then the primary antibody was rinsed by PBS, biotinylated secondary antibody (1:500) was added on the slides and incubated for 30 min. The second antibody was omitted and bound secondary antibody was detected by the 3,3’-diaminobenzidine (DAKO) chromogen by standard methods.The cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at room temperature for 15 min. Then the cells were incubated with primary antibody at 4°C overnight. Then the cells were washed with PBS and treated with secondary antibody at 37°C for 60 min. After PBS washing, the cells were stained with PADI to identify the nuclear. Then the cells were observed by a fluorescence microscope (Olympus IX70).A commercial ELISA kit  were used to identify the expression of IL-6 in conditioned medium collected from FLSs. The assay was performed accordingly to the protocol provided by the manufacturer. Means and standard deviations of concentrations in triplicate samples were compared by t-test.All data, expressed as mean ± SEM, were from at least three separate experiments. Data sets were analyzed by SPSS 18.0 software . Comparison was done with t test (unpaired or paired). All p values presented were two-sided. P<0.05 was considered to be statistically significant.


Polycystic ovary syndrome (PCOS) is associated with insulin resistance (IR) and compensatory hyperinsulinemia. IR is recognized as a major risk factor for the development of type 2 diabetes mellitus. However, few studies have investigated IR in women with PCOS and normal glucose tolerance. The objective of this study was to evaluate IR and β-cell function in women with PCOS and normal glucose tolerance. Additionally, we sought to evaluate the usefulness of oral glucose tolerance test (OGTT)-derived IR indices in lean women with PCOS.120, HOMA-F and the Stumvoll index, were calculated from an OGTT. Increased β-cell function was defined as>75th percentile for the HOMA-F in control women.We recruited 100 women with PCOS and normal glucose tolerance and 100 age- and BMI-matched women as controls. IR and insulin secretory indices, including the homeostasis-model assessment (HOMA)-IR, HOMA-M120, HOMA-F and lower values for the Stumvoll index than the controls . Women with PCOS and increased β-cell function showed lower Stumvoll index values than the matched controls (P<0.05). The HOMA-F was significantly associated with the HOMA-M120 and Stumvoll index when adjusted for age and BMI in a multiple regression analysis . The HOMA-M120 was positively correlated with triglycerides and free testosterone, and the Stumvoll index was negatively correlated with triglycerides and free testosterone in lean women with PCOS .Women with PCOS had higher values for post-load 2-hour glucose, fasting insulin, post-load 2-hour insulin, HOMA-IR, HOMA-MWomen with PCOS and normal glucose tolerance showed higher IR than controls matched for age, BMI, and β-cell function. β-cell function was increased in women with PCOS when compared to the matched controls, but not when the lean subjects were compared to the matched controls separately. Therefore, early evaluation of IR in women with PCOS and normal glucose tolerance may be needed. The HOMA-IR was calculated as the product of the fasting insulin level (mIU/L) and the fasting glucose level (mg/dL) divided by 405. The modified HOMA-My indices were calculated as follows: HOMA-My = Gy (mg/dL) × Iy (mIU/L)/405, where y and Iy indicate 120-minute glucose and insulin values from the OGTT, respectively [The 75-g OGTT was performed in the morning after an overnight fast. After 30 min of supine rest, venous blood samples were drawn at baseline, 90 min, and 120 min after the 75-g glucose load. Plasma glucose levels were measured via the glucose oxidase method , and insulin levels were measured by radioimmunoassay using a commercially available kit . Fasting total serum cholesterol, triglycerides (TG), and high-density lipoprotein (HDL) cholesterol levels were measured using an enzymatic assay on an automated analyzer . β-cell function was assessed by the homeostasis model assessment of β-cell function (HOMA-F): 20 × fasting insulin, mIU/L/. Increased β-cell function was defined as > 75120, Stumvoll index, and HOMA-F were logarithmically transformed to achieve a normal distribution. Quantitative variables were reported as the mean ± standard deviation. Variables that showed a skewed deviation were reported with medians and interquartile ranges. Between group differences were assessed by the unpaired t test or Mann-Whitney U-test. The Spearman’s rho correlation coefficient was applied to assess the correlation between the insulin sensitivity indices and the hormonal and metabolic features of PCOS. Multiple linear regression analyses were performed to determine the independent association between HOMA-F and the insulin resistance indices. A P value < 0.05 was considered significant.Statistical analyses were performed using the SPSS 18.0 software package for Windows . The Kolmogorov-Smirnov statistic was used to analyze the continuous variables for normality. The levels of the HOMA-IR, HOMA-M120, HOMA-F and lower value of Stumvoll index values than the corresponding controls    . Lean wo < 0.05) .P < 0.05) . Lean women with PCOS and increased β-cell function showed a higher value for HOMA-M120 and lower value of Stumvoll index values than lean controls with increased β-cell function   . The HOM < 0.05) .120 (β = 0.321) and negatively associated with the Stumvoll index (β = -0.287) when adjusted for age and BMI . In lean women with PCOS, the HOMA-F was positively associated with the HOMA-M120 (β = 0.272) at a marginally significant level when adjusted for age and BMI   .120 could be a simple tool to assess IR and was correlated with hyperandrogenemia and hypertriglyceridemia in lean women with PCOS.In our study, women with PCOS who had normal glucose tolerance showed higher IR than age-, BMI-, and β-cell function-matched controls, and these differences held for lean women with PCOS compared to matched controls. β-cell function was increased in all women with PCOS compared to matched controls; however, β-cell function was not increased in lean subjects when compared alone. As described previously , the HOMIR was detected in over half of women with PCOS using fasting glucose and insulin . Because120 index has provided a very simple measure of IR in lean white women with PCOS [120 is a useful index for evaluating IR in Asian women with PCOS. Lean women with PCOS, even when β-cell function is matched, showed higher values for the HOMA-M120, but not the HOMA-IR, compared to matched controls in our study. The HOMA-M120 index was obtained from the calculation equation including post-load 2-hour glucose and insulin, however, the HOMA-IR index was obtained from the calculation equation including fasting glucose and insulin. Further studies are needed to evaluate the mechanism of IR in lean women with PCOS.There are many methods for evaluating IR. The glucose clamp technique is regarded as the best available standard for the measurement of insulin action. However, the cost and time demands of the glucose clamp have led to the development of simpler methods to measure IR, such as the HOMA calculation . Among tith PCOS . Here we120 was positively correlated with hypertriglyceridemia in lean women with PCOS but the HOMA-IR was not. Approximately 70% of women with PCOS have dyslipidemia [120 could be a useful tool for predicting IR as well as hyperandrogenemia and hypertriglyceridemia, which are important characteristics of PCOS.Previously, IR was reported to be significantly related to free testosterone levels . In the ipidemia . TherefoPCOS is associated with profound IR as well as defects in insulin secretion . HoweverThis is the first study to evaluate IR between women with PCOS and β-cell function-matched controls. The strength of our study is the use of well-matched age and BMI controls, which enabled us to perform group comparisons without bias. However, there are several limitations to our study. Ethnicity and age affect the prevalence of IR in women with PCOS . Therefo120 could be a simple and reliable tool to assess IR in lean women with PCOS.Women with PCOS and normal glucose tolerance showed higher IR than age-, BMI-, and β-cell function-matched controls. Therefore, early evaluation of IR, even in lean women with PCOS and normal glucose tolerance, might be needed. Our data indicate that the HOMA-M


Parkinson’s disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect. While llifespan . These flifespan  or the 6lifespan . In addilifespan .O-acyltransferase (GOAT). This acylation step has long been considered necessary for its binding to the ghrelin receptor, although some recent studies show that des-acyl ghrelin, which lacks the acyl group, can function via the ghrelin receptor [During the early stages of CR, acylated ghrelin levels are significantly elevated ,17. Ghrereceptor ,20. In treceptor ,22. Ghrereceptor . Surprisreceptor ,25,26,27d-Lys-3-GHRP-6 abolished the protective effects of ghrelin in this model [Ghrelin enhances dopaminergic neuron firing in the SNc and increases dopamine release and turnover in the striatum ,29. Studis model . In addiis model . This fiis model ,32, whicis model . In a seis model . These fis model , the enhPD includes different molecular mechanisms  that contribute to disease progression. So far, only symptomatic treatment is available for patients. One of the possible reasons for the inability to translate preclinical findings to patient treatment is the poor diversity of animal models used during drug validation . For thiThe proteasome inhibition model of PD is a relatively new toxin-induced model, based on the intracerebral administration of proteasome inhibitors, such as lactacystin (LAC), to the nigrostriatal pathway . This apApart from the MPTP and rotenone models, which are strongly dependent on mitochondrial inhibition, less is known regarding the putative protective effects of CR against neurotoxins that trigger alternative pathways. Several studies showed that caloric restricted animals rescue 26S proteasome activity in the brains of aged rats and display increased autophagy in cortical neurons through the ghrelin receptor and also in other tissues ,41,42. PFinally, we studied whether, under physiological conditions, genetic deletion of the ghrelin receptor induces the degeneration of the nigrostriatal tract. Our study showed that CR protects against lactacystin-induced DA-ergic degeneration, independent of the ghrelin receptor.p > 0.05) (p > 0.05), independent of age (No difference was detected in the number of TH+ neurons in the SNc of adult (3–4 months) or aged (18–22 months) ghrelin receptor KO mice and their WT littermates ( > 0.05) A,B. Alsot of age C,D.p = 0.06 in sham mice, p = 0.075 in LAC lesioned mice)  A, and bled mice) C,F. It sp < 0.01) and in AL-fed ghrelin receptor KO mice (p < 0.01) (p < 0.01) (p > 0.05), and a significant increase in TH+ neuron survival was visible compared to the LAC AL group (p < 0.01)  A–C. Furt < 0.01) D. Intere < 0.01) D. Overalp < 0.0001) (p < 0.001) (p < 0.05)  E–G, and < 0.001) G. At 4 w < 0.05) G. Again,As previously reported, our present study confirms that intranigral LAC injection in mice in a dose of 3 µg significantly induces nigral DA-ergic cell loss and decreases striatal DA concentration . We showWhen using intermittent fasting, the neuroprotective effect, as was seen with CR,was not that clear as only one study could prove that CR reduced DA-ergic cell loss after injecting MPTP or 6-OHDA ,47,48. HUsing a CR regimen that was reported to elevate plasma ghrelin levels  we foundCR restriction induces the expression of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) that could be involved in protecting the nigrostriatal DA pathway against toxic stimuli . InteresAnother possible mechanism of action of CR is the sirtuin 1-autophagy pathway. Previous studies showed that CR can induce autophagy through enhancement of AMPK and Sirtuin 1 activity, thereby decreasing protein aggregation and increasing longevity ,53. AlthAn alternative, but not mutually exclusive, hypothesis is that the increase in plasma ghrelin levels observed after CR in our group of mice might have been insufficient to stimulate ghrelin receptor activation in the SNc. This could explain the lack of involvement of the ghrelin receptor following CR, especially as the protective effects of ghrelin in the MPTP model were found to be dose-dependent . AlthougAnother possibility that can explain why we do not see a difference in neuroprotection between ghrelin receptor WT and KO mice is that des-acyl ghrelin induces neuroprotection in the LAC mouse model. A previous study showed that in vitro des-acyl ghrelin inhibits oxygen-glucose deprivation-induced cell death in cultured neurons . In vivoOf note, ghrelin has recently been shown to stimulate the function of the proteasome pathway in cellular models of Alzheimer’s disease . WhetherAs an important control experiment we checked for possible spontaneous neurodegeneration by ghrelin receptor deletion; nevertheless, we also did not see changes in TH+ neurons in adult or aged ghrelin receptor KO mice versus their WT littermates. Thus it seems that the absence of the ghrelin receptor does not induce nigral degeneration.Together with our study, it is clear that CR shows a neuroprotective effect in several animal models with different mechanisms of action; hence this implies that CR remains an interesting path to further investigate and thereby unravel which mechanisms are behind this neuroprotective effect. Unfortunately, we were not able to confirm in this model that the CR-induced neuroprotective effect is mediated via the ghrelin receptor, making it not the main factor involved in the CR-induced neuroprotective effect in the LAC mouse model. As the neuroprotective effect of ghrelin in the MPTP mouse model could not be reproduced in the LAC mouse model, it remains dubious whether the ghrelin axis could be a potential target for future treatment in PD.All experiments were carried out according to the National guidelines on animal experimental research and the procedures were approved by the Ethical Committee for animal experiments of the Vrije Universiteit Brussel . Male adult (3–4 months) and aged (18–22 months) ghrelin receptor WT and ghrelin receptor KO mice on a mixed 129Sv/Evbrd(LEX1)/C57BL/6 background were used for this study. The ghrelin receptor KO mice were created by Janssen Pharmaceutica  in collaboration with Lexicon Genetics, Inc , as previously described . GenotypA group of adult ghrelin receptor WT and ghrelin receptor KO littermates and a group of aged ghrelin receptor KO and WT littermates were fed AL and were sacrificed without any further intervention (no surgery) to investigate the effect of the genetic ablation of the ghrelin receptor on the neurodegeneration of the nigrostriatal pathway.Before assigning the mice to their feeding regimen, the adult mice were single housed and their food intake was measured for 5 days. Starting 21 days prior to LAC or sham injection, mice undergoing CR received 70% of normal food consumption, based on their individually-calculated daily food intake. Tap water was available AL for both the CR- and AL-fed groups. Mice were weighed and fed daily around 2 h before the onset of the dark cycle. Blood glucose (BG) was measured every 2 days prior to feeding. Mice remained on the feeding regimen until the termination of the experiment; 28 days in total. On the 28th day, body weight (BW) and BG were measured immediately before sacrifice.On the 21st day of the feeding regimen, mice underwent surgery as previously described . BrieflyOne week after surgery, all mice were sacrificed by cervical dislocation following sedation and trunk blood was collected in ethylenediaminetetraacetic acidEDTA tubes pretreated with Pefabloc SC . The blood was centrifuged and the collected plasma (supernatant) was treated with 0.05 N HCl. Plasma samples were used for the determination of acylated ghrelin using an ELISA kit according to the manufacturer’s instructions .g at 4 °C and the supernatants were collected and 1:5 diluted in 0.5 M acetic acid. For the determination of total DA content in the ipsi- or contralateral striatum, 20 µL of the mixture was analyzed using narrow-bore  liquid chromatography with electrochemical detection, as previously described [After cervical dislocation, the brains were rapidly removed and the striata were isolated on an ice-cold petri-dish. Next, both the ipsi- and contralateral striatum were weighed and homogenized separately (30 s) in 400 µL antioxidant solution containing 3,4-dihydroxybenzylamine (1 µg/mL) as an internal standard. The homogenates were centrifuged for 20 min at 10,000× escribed . Results3. Nigral sections were sampled throughout the entire rostrocaudal extent of the SNc (2.92 mm until 3.64 mm posterior to bregma) to investigate the presence of TH-expressing neurons in the ipsi- and contralateral SNc. During the staining, all the rinsing steps and all the dilutions were performed in Tris-saline. On the first day of the staining, 3% H2O2 was administered followed by a blocking step with normal goat serum  and overnight incubation with rabbit anti-TH antibody . On the second day, sections were processed by the avidin-biotin method, using the Vectastain ABC kit , and the glucose oxidase-diaminobenzidine-nickel method was used for visualization. All sections were air-dried, cleared in xylene, and coverslipped with Dystirine Plasticer Xylene (DPX). Photomicrographs were made of the stained sections and cell counts of the SNc were performed using ImageJ software . The number of TH+ neurons was counted in six serial sections throughout the entire rostrocaudal extent of the SNc (2.92 mm until 3.64 mm posterior to bregma) (staining of 1 section per 3 sections) by a scientist blinded to the genotype and the feeding regimen. This total number was then multiplied by three.After cervical dislocation, the caudal part of the brain was removed and post-fixed for three days in freshly prepared 4% paraformaldehyde. Free-floating sections of 40 µm were sliced with a vibratome and were stored at 4 °C in 10 mM PBS + 1.5 mM NaNt-test to compare the nigral TH+ profiles and striatal DA content in adult and aged ghrelin receptor WT and KO mice. For all other comparisons, a two-way ANOVA followed by Sidak’s multiple comparisons test was used. The α-value was set at 0.05.Statistical analysis was performed using GraphPad Prism 6.0 software. Data are expressed as mean ± SEM. We used a According to our findings, CR has a neuroprotective effect in the LAC mouse model for PD. Our finding is in line with the previous reports that showed a neuroprotective effect of CR in the MPTP and 6-OHDA models ,12,33. H


Wilson’s disease (WD) is an autosomal recessive disorder. It is characterized by toxic accumulation of copper mainly in the liver and brain but also in cornea and kidney due to a defect in biliary excretion of copper. The hepatic manifestation of WD is diverse and may include asymptomatic elevation of aminotransferase, chronic hepatitis, cirrhosis, or acute/fulminant hepatic failure. Characteristic of acute hepatic failure in WD is concomitance of acute intravascular hemolytic anemia. Acute intravascular hemolytic anemia and thrombocytopenia in WD may be interpreted as a feature of Hemolysis, Elevated Liver Enzymes, Low Platelet Count (HELLP) syndrome besides acute liver failure. The differential diagnosis may be very difficult. Here, WD in pregnancy presenting with clinical symptoms of HELLP syndrome and developing acute liver failure in postpartum period is discussed. Carrier frequency is 1/90. The gene responsible for the disease is ATP7B, located on chromosome 13. It is most commonly seen in the second-third decades of life. In more than 80% of patients, the symptoms of the disease are observed in the first three decades. The symptoms of WD-related fulminant hepatic failure are particularly seen in female patients below the age of 40 years. Clinical findings are quite various and include hepatic and neuropsychiatric symptoms. The hepatic manifestation of WD is diverse and may include a clinical presentation that varies from asymptomatic elevation of aminotransferase, chronic hepatitis and cirrhosis, to acute/fulminant hepatic failure.Wilson’s disease (WD) was first identified by Kinnear Wilson in 1912. The incidence of the disease is reported as 1/30000-1/100000 births. Acute intravascular hemolysis and thrombocytopenia in a pregnant woman known to have WD may be interpreted, together with acute liver failure, as a feature of HELLP syndrome. The differential diagnosis may be difficult however. HELLP syndrome is seen in 0.1-0.6% of pregnant women, particularly in the second trimester (in 70% of cases) and in the postpartum period (in the first 1-2 days). The laboratory criteria for HELLP syndrome were first defined in 1986. These are hemolytic findings (bilirubin increase >1.2 mg/dL), abnormal liver functions (AST>70 IU/L), reduced thrombocytes of less than 100x109/L. Endothelial damage or dysfunction causes abnormal expression of inflammatory mediators and an activation and aggregation of thrombocytes. This leads to the fragmentation of red blood cells, hemolysis and microangiopathic anemia. In addition, an abnormal activation of thrombocytes and thrombocytopenia arising from increased use are observed. Here, a case of WD in pregnancy, presenting clinical manifestations of HELLP syndrome concomitant with acute liver failure developed during postpartum period is discussed.The acute liver failure in WD is concomitant with acute intravascular Coombs-negative hemolytic anemia and, in some patients, may be manifested as the first symptom of the diseaseth week of spontaneous pregnancy, gravida 1, parity 0, was admitted at our obstetrics clinic with the start of labor. The patient’s pregnancy controls were taking place at our obstetrics outpatient clinic as from the 31st week of pregnancy. An abdominal ultrasonography performed when the patient was seventeen years old revealed a cirrhotic liver. The biochemical test results showed a ceruloplasmin level of 10.1mg/dl (25.0-45.0 mg/dl), free copper levels of 71μgr/dl , a urine copper level of 120 μgr/24 hr , upon which a liver biopsy was performed leading to a diagnosis of Wilson’s disease. From an investigation into the patient’s family history, it was learned that a sibling had undergone a liver transplant due to Wilson’s disease. Also followed up by the gastroenterology outpatient clinic, the patient was being treated with 300 mg 3x1 of Metalcaptase (D-penicillamine) and 2x1 p.o. of Zinco (50 mg zinc) throughout her pregnancy but she stopped taking this treatment for the last three days. On the patient’s examination, blood pressure was 140/90 mmHg and in complete urinalysis, proteinuria was found to be at +++ level. Furthermore, the following results were obtained in the blood tests; AST: 45 U/L (5-40 U/L), ALT: 20 U/L (5-40 U/L), ALP: 216 U/L (40-150 U/L), LDH: 406 U/L (125-243 U/L), amylase: 47 U/L, total bilirubin: 1.2 mg/dL (0.2-1.2 mg/dL), direct bilirubin: 0.64 mg/dL (0-0.5 mg/dL), total protein: 4.7 g/dL (6.4-8.3 g/dL), albumin: 2 g/dL (3.5-5 g/dL), thrombocytes: 98x109/L (150-300x109/L). The patient was diagnosed with pre-eclampsia. The additional pelvic examination revealed pronounced cephalopelvic disproportion and a cesarean section was therefore carried out. The patient delivered a live 3050-gram male infant in cephalic presentation with an APGAR score of 7-9. The patient was followed up on the hospital ward in the postpartum period. A change in the laboratory tests was observed at 24 hours postoperatively. The clinical results were as follows: AST: 50 U/L (5-40 U/L), ALT: 19 U/L (5-40 U/L), total protein: 4.1 g/dL (6.4-8.3 g/dL), albumin: 1.6 g/dL (3.5-5 g/dl) and thrombocyte values: 89x109/L (150-300x109/L). A gastroenterological evaluation of the patient was ordered but the patient was discharged on the 2nd day postoperatively on her own request. On the third postoperative day, the patient was admitted to the emergency room with the complaints of widespread swelling in the abdomen, vulva and legs. Physical examination revealed massive vulvar edema, pretibial edema in the lower extremities, and the abdomen was distended  and a drop in blood values (Hb): 10.7 g/L , hematocrit (Hct): 30.1% (37.0-44%), RBC: 3.30x106/mcrL (3.83-5.08 106/mcrL), PLT: 40x109/L (150-300x109/L). The values in the coagulation profile were; prothrombin time : 1.29 (08-1.2), Aptt: 41.1s (26.6-40.0), fibrinogen: 142 (180-350), D-dimer: 4871 (50-228). Kidney function tests were normal. When a full recovery could not be seen in the clinical and laboratory findings with the ongoing treatment and because of the need for a liver transplantation in the long term, the patient was referred to a hospital that had an organ transplantation department. The patient has been waiting her turn for a liver transplantation for the last two months and the current treatment continues, .A 24-year-old patient in the 37istended . Blood p. The symptoms of WD comprise hepatic findings at a rate of 40-70%,10 and neuropsychiatric findings at a rate of 50%. Hepatic symptoms are more pronounced in children while neurological and psychiatric symptoms are more common in adults. In this case however the patient presented with hepatic symptoms and no neuropsychiatric symptoms were observed.WD is an autosomal recessive disease. It is characterized by toxic accumulation of copper, mainly in the liver and brain but also in the cornea and kidney, due to a defect in the biliary excretion of copper. Furthermore, it is not uncommon for a woman in this category to be suffering from amenorrhea, oligomenorrhea, irregular menstruation, and repeated miscarriages. In symptomatic cases, hormonal changes related to liver dysfunction disrupt fertility. The diffusion of non-ceruloplasmin-bound copper from plasma into tissues may affect ovarian follicular aromatase activity. Earlier studies have reported healthy full-term pregnancies in treated patients, but miscarriage and pregnancy loss rates show an increase in untreated cases. Here, we report a case with spontaneous pregnancy at full term with no previous treatment for infertility.Neurological and psychiatric adversities in patients with WD preclude the possibility of marriage. D-penicillamine increases excretions of copper in the urine by creating non-toxic copper-penicillamine compounds; it is the first preference for treatment,2. Patients who do not tolerate this treatment are given trientine as an alternative. Zinc competes with copper and prevents intestinal copper absorption, proving to be an effective medication in long-term maintenance of the disease. In the present case, the treatment option of penicillamine  was chosen.The aim of treatment is the removal of copper from the body and the prevention of copper absorption through diet. In addition, treatment should continue in pregnancy in order to avert the development of hemolysis, liver dysfunctions and liver failure. Dosage should be kept at the lowest effective level and the dose should be reduced in last trimester to 25-50% of the dose administered before the pregnancy in order to avoid interfering with wound-healing. We believe that in the present case, the patient was treated with penicillamine throughout the entire pregnancy and stopping the drug in the last three days triggered the acute liver failure. There is no other publication in the literature reporting development of acute liver decompensation in such a short time after the cessation of treatment. In fact, Shimono et al. reported a case of pregnancy with no problems during treatment with D-penicilline which yielded two births. Treatment was stopped at the third child of the same patient and acute liver failure caused by hemolytic crisis stemming from the accumulation of copper in the body occurred after the delivery. We also believe that acute liver failure seen in the postpartum period in the present case was caused by the same mechanism.D-penicillamine is recommended for treatment during pregnancy since it is well-tolerated and safe for both mother and child. A smaller dose is used in the treatment of WD and it is rapidly eliminated from the body after binding with copper by chelation and its level decreases even lower. It is because of this reason, treatment with penicillamine during pregnancy has been reported to be safe, this value would have been much higher in acute liver failure(22). At the same time, this patient had leukocytosis, an elevated D-dimer and reduced serum albumin. Although these findings are less typical in HELLP syndrome,,23 the proteinuria found in the spot urine test (+++) does suggest HELLP. Elevations in serum aminotransferase in WD are less than 10-fold, as in this case. Moreover, there were also some anomalies found in the coagulation test; there were prolongation of INR and APTT values. In addition, the fact that blood pressure measured as 140/90 mmHg indicates WD together with HELLP syndrome. The normal kidney function tests of the patient steered us away from hepatorenal syndrome. On the other hand, the patient progressed into acute liver failure in the postpartum period.In this report, a case of WD in pregnancy, presenting with the clinical manifestations of HELLP syndrome concomitant with postpartum acute liver failure has been discussed. Since no microscopic blood analysis was performed, a direct microscopic imaging of hemolytic anemia was not carried out. At first presentation with HELLP syndrome, total bilirubin was 1.2 mg/dl and direct bilirubin was 0.64 mg/dl; in the period when the liver failure developed, total bilirubin was found to be 3.00 mg/dl. As it was in this case, even though total bilirubin is normal or minimally increased (1-2 mg/dL) in HELLP syndromeTo conclude, the main objective in following up cases of concomitant WD and pregnancy is to evaluate maternal and fetal effects and to control the disease using the lowest possible dosage of drug. A multidisciplinary approach and the close monitoring of high-risk pregnancies will lead to similar positive outcomes in the general population. WD is a chronic liver disease that allows a normal life if diagnosed and treated early. It is a condition that responds to treatment, even when cirrhosis has been diagnosed, but needs life-long treatment and care. Treatment should continue over the life span and should only be stopped when a liver transplant has been performed. Even a short interruption in the treatment of only three days in this case has the potential of speeding up the process of acute hepatic failure and the development of decompensation.


The targeting and long-term imaging of the Golgi apparatus have been realized vial-cysteine functionalized nanoprobes. l-cysteine. l-Cysteine-rich chiral carbon quantum dots (LC-CQDs), which have the benefits of a high Golgi specificity from l-cysteine and excellent photostability and biocompatibility from the CQDs, are proven to be highly suitable for long-term in situ imaging of the Golgi. Investigation of the mechanism showed that free thiol groups and the l-type stereo configuration of LC-CQDs are essential for specific targeting of the Golgi. With the aid of the as-prepared LC-CQDs, the dynamic changes of the Golgi in the early stage of viral infection were visualized. The Golgi targeting and imaging strategy used in this work is beneficial for Golgi-targeted drug delivery and early diagnosis and therapy of Golgi diseases.The Golgi apparatus is an essential subcellular organelle. Targeting and monitoring the Golgi change at the single-cell level over a long time scale are critical but are challenges that have not yet been tackled. Inspired by the precise Golgi positioning ability of galactosyltransferase and protein kinase D, due to their cysteine residues, we developed a method for long-term Golgi imaging. Fluorescent molecules, carbon quantum dots (CQDs) and silica nanoparticles could target the Golgi when they are modified with  With the development of the membrane fusion method using N-[7-]-6-aminocaproyl sphingosine (NBD C6-ceramide) for exploring the sphingolipid transport and metabolism mechanism,5 ceramide analogues have become one of the most widely used commercial dyes for labeling the Golgi.6 Due to the lack of specificity of ceramide analogues, however, the plasma membranes as well as the mitochondria are also labeled eventually besides the Golgi apparatus. Other Golgi stain approaches,8 using antibodies, for example, also lack long-term imaging ability, making it difficult to observe real-time changes of the Golgi at the single-cell level under continuous laser excitation. Therefore, it is highly desirable to design and develop new optical probes for the Golgi with a prolonged imaging ability.Subcellular targeting strategies have redefined the frontier of life processes as well as drug design.9 have been found to be physicochemically and photochemically stable without photobleaching, making them a star family in the biosensing and bioimaging fields.13 However, bare CQDs are usually weakly fluorescent and it is hard to attach functional groups on the surface.15 Regardless of continuous efforts to circumvent the problems,16 challenges still remain to engineer CQDs with desirable biosensing and labeling properties, such as a high quantum yield and preferable selectivity for the long-term and real-time monitoring of targets of interest.17Fluorescent carbon quantum dots (CQDs), since their first discovery in 2004,in situ imaging of the Golgi apparatus. It has been reported that galactosyltransferase and protein kinase D are capable of anchoring in the Golgi apparatus via their cysteine residues or cysteine rich domain,19 which inspires us to combine the principle of the Golgi localization of proteins and carbon nanotechnology to develop an optical probe for Golgi targeting and imaging. We synthesize novel fluorescent CQDs with abundant cysteine residues and an l-type spatial structure using a pyrolysis method with citric acid and l-cysteine as the carbon sources and controlling the pyrolysis temperature. The as-prepared LC-CQDs exhibit excellent long-term Golgi targeting and imaging capabilities that could be attributed to their high quantum yield (68%) and photostability as well as their good biocompatibility. This dependence of the targeting of the Golgi on l-cysteine is further proven by using cysteine modified fluorophores and silica nanoparticles. This study provides an effective method for Golgi targeting and imaging over a long time scale, which may be applied in drug-delivery and therapy, as well as in evaluating disease progression occurring within the Golgi.Herein, we report a novel type of CQD for the long-term l-cysteine is well preserved on the surface of the LC-CQDs, as indicated by the vibrational frequency  of the free thiol groups  display opposing Cotton effects in the range of 200–400 nm, reinforcing the existence of the chirality in both LC-CQDs and DC-CQDs. Thus, it can be inferred that the chirality of the as-prepared LC-CQDs and DC-CQDs is transferred from cysteine to the CQDs.The as-prepared LC-CQDs eventually are 8.5 ± 3.5 nm in diameter and are highly fluorescent with an absolute quantum yield of 68% . The fre Fig. S11. The devλmax) of 420 nm, giving an absolute quantum yield of 68%. The strong fluorescence emission of the LC-CQDs is attributable to the co-doping effect of N and S.23 Significantly, the LC-CQDs exhibit superior photostability compared to the FITC dye, fluorescein and the CdTe QDs (regarded as photostable fluorescent labels), as LC-CQDs display excellent fluorescence properties. As N-acetylgalactosaminyltransferase-GFP (Golgi-GFP) and Bodipy ceramide, the two frequently adopted probes for Golgi-staining. Obvious fluorescence in the Golgi is observed (–1). The fluorescent area matches very well with those of Golgi-GFP and Bodipy ceramide  with observed  after 4 ceramide  with a Pceramide . Similarceramide . The intns-Golgi , and thens-Golgi .in situ imaging of the Golgi, different properties, such as imaging duration, biocompatibility and photostability, were evaluated. The cell number increases from four to seventeen, due to cell proliferation (–1) does not affect the quantity of secreted proteins, glycoproteins and apoptosis proteins porphine (TCPP) is linked with different numbers of l-cysteine residues. Unfunctionalized TCPP is found to spread over the whole cells, while single cysteine functionalized TCPP colocalizes well with Golgi-GFP  with abundant cysteine residues. The LC-CQDs exhibit unique properties, including a high quantum yield (68%), high photo-stability, as well as favorable biocompatibility. More importantly, the LC-CQDs exhibit a distinctive capacity for long-term in situ Golgi imaging via the l-type stereo structure and free thiol groups. Due to their long-term in situ Golgi imaging ability, the LC-CQDs are able to trace the real time process of intracellular injury, such as viral infection, by monitoring the morphological changes of the Golgi apparatus. Thus a transition stage from the living state to the death of the cells during the process of viral infection has been observed, providing an innovative mode of investigating diagnostically relevant changes in the early stages of viral infection. Furthermore, the exploration of cysteine for Golgi targeting provides an innovative subcellular targeting strategy for drug design and delivery, and thus illustrating that functional cellular CQDs that can target subcellular organelles such as Golgi body show high promise in the explorations of cellular activities of typical subcellular organelles.We have successfully demonstrated an approach to prepare chiral carbon quantum dots (l-cysteine and citric acid (CA). CA monohydrate  was at first placed in a 50 mL flask and heated at 200 °C for 20 min, and then l-cysteine  was added. After stirring with a glass rod, the yellow mixture was kept at 200 °C for 60 min until a dark product was obtained. 2 mL of ultra-pure water was added to the mix of carbon quantum dots with cysteine and heating was continued if the yellow mixture did not turn dark. The dark product was finally neutralized with KOH solution . The LC-CQDs were collected by removing the large particles through filtering using 0.22 μm membranes, and then dialyzing against ultra-pure water for 48 h to remove small organic molecules and salts . Other carbon quantum dots were synthesized by replacing the l-cysteine with d-cysteine, l-alanine, S-methyl-l-cysteine, thioacetamide and a gold–cysteine complex .The LC-CQDs were prepared by directly pyrolysing N,N′-disuccinimidyl carbonate  and N,N-diisopropylethylamine . After another 12 h, the DSC-functionalized silica nanoparticles were collected and washed with acetone. Finally, the DSC-functionalized silica nanoparticles were redispersed in 8 mL l-cysteine (Sigma-Aldrich) complex solution  for 4 h. The l-cysteine functionalized fluorescent silica nanoparticles (LC-F-SiO2) were collected by centrifugation at 12k rpm and washed with ultra-pure water. Some large products could be removed by centrifugation at 4k rpm.Methanol (10 mL), ultra-pure water (3 mL), and concentrated ammonia (0.7 mL) were mixed at 30 °C under stirring. Tetraethyl orthosilicate (200 μL) was then added to the solvent mixture followed by the addition of a fluorescent complex , and 4.8 μL 3-aminopropyltriethoxysilane (APTES). The fluorescent complex solution should be mixed and stored at 4 °C for one day before the synthesis process). After 4 h, the products were collected by centrifugation at 12k rpm and washed with ethanol. By adjusting the amount of concentrated ammonia used under the same conditions, the particle sizes of the silica nanoparticles were tuned to 40 nm , and 80 nm . Subsequently, the as-prepared silica nanoparticles were dispersed in 15 mL of ethanol followed by the addition of 200 μL APTES. After 12 h, the amine-functionalized silica nanoparticles were collected and washed with ethanol. The amine-functionalized silica nanoparticles were then redispersed in 15 mL acetone followed by the addition of –1 penicillin G and 100 μg mL–1 streptomycin sulphate in a CO2 incubator at 37 °C. One day prior to adding the LC-CQDs, the cells were seeded on 35 mm glass-bottom dishes (NEST. Corp.). The cells were then incubated with the LC-CQDs  overnight. After washing three times with phosphate buffered saline (PBS), the cells were observed under a confocal microscope. Fluorescein, fluorescein–cysteine-1, fluorescein–cysteine-2, TCPP, TCPP–cysteine-1, TCPP–cysteine-2, F-SiO2 and LC-F-SiO2 were incubated and observed under the same conditions. Fluorescein–cysteine-1, fluorescein–cysteine-2, TCPP, TCPP–cysteine-1, and TCPP–cysteine-2 were designed by our group and synthesized by Sangon Biotech (Shanghai) Co., Ltd.In the cellular experiments, cells were cultured in RPMI 1640 (Hyclone) media supplemented with 10% foetal bovine serum , 100 U mL5-ceramide (Thermo Fisher Scientific) or NBD C6-ceramide (Thermo Fisher Scientific) can be administered to cells as a complex with bovine serum albumin (BSA). The cells were washed until they were free of culture media and incubated with Bodipy TR C5-ceramide (5 μM) for 30 min at 4 °C. The cells were then washed, incubated in 2% BSA solution for 90 min at 25 °C, and finally observed under a fluorescence microscope. CellLight® Golgi-GFP (Thermo Fisher Scientific) was added to the cells, incubated overnight, and the cells were ready to image in the morning.Bodipy TR C6-ceramide, F-SiO2, LC-F-SiO2, fluorescein, fluorescein–cysteine-1, and fluorescein–cysteine-2 were excited at 470–490 nm and detected with a barrier filter BA 510–550 nm. BODIPY TR C5-ceramide, TCPP, TCPP–cysteine-1 and TCPP–cysteine-2 were excited at 530–550 nm and detected with a barrier filter BA 575–625 nm.All the carbon quantum dots were excited at 360–370 nm or 405 nm and detected with a barrier filter BA 420–460 nm. Golgi-GFP, NBD CThe HEp-2 cells used in this experiment are the laryngeal cancer cell lines. The respiratory syncytial virus (RSV) used in this experiment is effective at infecting HEp-2 cells. In order to monitor the morphology changes of the Golgi during the viral infection, we selected Hep-2 cells as the host cells.n = 5).The data in the main text and ESI–1 Au-doped LC-CQDs for 48 h, washed, and fixed in 1% glutaraldehyde. The cells were then washed with 0.1 M PBS buffer and treated with 0.5% OsO4 for 1 h at 4 °C. After rinsing with ultra-pure water, the cells were stained with saturated uranyl acetate in 70% ethanol for 24 h at 25 °C. The cells were then dehydrated in ethanol and embedded in Epon. Thin sections were obtained with an ultramicrotome. Electron micrographs were obtained with an electron microscope (Transmission Electron Microscope Hitachi-7500). Control samples were treated as described above, except that they were not incubated with LC-CQDs.Cells grown in cell culture flasks were incubated with 1 mg mLThe cells were fixed in 4% paraformaldehyde for 20 min at 25 °C and permeabilized with 0.1% Triton X 100 for 2 min at 25 °C. The primary antibodies were diluted 1/200 and incubated with samples for 1 h at 37 °C. The secondary antibody was the goat polyclonal secondary antibody to Rabbit IgG–H&L (Cy3®) used at a 1/500 dilution for 1 h. 2% BSA was used for the blocking steps. All antibodies were purchased from Abcam.2 incubator. The morphology of the Golgi apparatus of the same cell was observed at different times.Virus samples were diluted with RPMI 1640 and then introduced to the cells. The Golgi apparatus of the cells had been labelled by LC-CQDs before the addition of the virus. After incubating with RSV for 2 h at 37 °C, the infected cells were cultured in RPMI 1640 supplemented with LC-CQDs and 2% FBS in a 5% COHEp-2 cells used in this experiment were the laryngeal cancer cell lines. The respiratory syncytial virus (RSV) used in this experiment was effective at infecting HEp-2 cells.The absolute photoluminescence quantum yield was measured using Quantaurus-QY .